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Abstract

Previous studies have demonstrated that compared with more buoyant LDL, dense LDL (D-LDL) is more susceptible to oxidation and less readily protected from oxidation by antioxidant enrichment. However, diets enriched in monounsaturated fatty acids (MUFAs) appear particularly effective in protecting D-LDL from oxidation. We therefore evaluated in 12 non–insulin-dependent diabetes mellitus subjects the effects of supplementation with α-tocopherol (1600 IU/d) and probucol (1 g/d) alone and in combination with an MUFA-enriched diet on LDL and LDL subfraction susceptibility to oxidation and monocyte release of superoxide anion. Subjects received either α-tocopherol or probucol for 4 months, and during the fourth month both groups also received an MUFA-enriched diet. α-Tocopherol levels were significantly increased in LDL and LDL subfractions (P<.05) after 3 months of supplementation. MUFA-enriched diets led to further increases in α-tocopherol in LDL fractions in the α-tocopherol group as well as in those receiving probucol. In the α-tocopherol–supplemented group, lag times were increased significantly (1.6- to 2.0-fold) for all LDL fractions, although the absolute increase was least for D-LDL. Although probucol supplementation increased lag times of LDL and LDL subfractions three- to fourfold, D-LDL was still more readily oxidized. In both the α-tocopherol– and probucol-supplemented groups the benefit of adding MUFA-enriched diets was greatest for D-LDL, with further increases in lag time of 26% and 18%, respectively. Neither antioxidant supplementation nor the addition of an MUFA-enriched diet reduced unstimulated or phorbol ester–stimulated monocyte superoxide anion production. These data demonstrate the markedly different effects that antioxidants and diet may have on different LDL subfractions, which may be particularly important in individuals with non–insulin-dependent diabetes mellitus, who frequently have increased amounts of D-LDL.

Recent advances in our understanding of the pathogenesis of coronary heart disease may help to provide explanations for the increased risk for atherosclerosis present in NIDDM. The current hypothesis is that after leaving the antioxidant-replete environment of the circulation and entering the artery wall, LDL is oxidized by artery wall cells via mechanisms that are not yet fully understood. Once oxidized, LDL takes on a number of properties that makes it more atherogenic12 and that appear to vary with the extent of LDL modification.34567

A major determinant of LDL oxidation in vivo, and therefore an important first step in the development of atherosclerosis, is the inherent susceptibility of LDL to oxidation. The susceptibility of the LDL particle to oxidation is dependent in part on its content of polyunsaturated fatty acids,891011 which are the primary substrates of lipid oxidation, as well as the content of endogenous antioxidants, primarily vitamin E, and possibly β-carotene and ubiquinol12131415 and supplemented antioxidants.16171819 To date these compositional factors have not consistently differed between LDL samples isolated from diabetic and control subjects and thus are less likely explanations for differences in LDL oxidation and rates of atherosclerosis between these two groups. The susceptibility of LDL to oxidation also appears related to LDL particle size, although this likely reflects structural and compositional differences among LDL subfractions. Several epidemiological studies have shown LDL of greater density (and reduced size) to be associated with an increased risk for coronary heart disease,2021 although its independent contribution to atherosclerotic disease remains in question. D-LDL can enter the artery wall more readily than larger lipoprotein particles,2223 and once in the subendothelial space, D-LDL may be preferentially retained.24 Most importantly, D-LDL is more susceptible to oxidation.252627 This combination of more rapid entry into the artery wall, prolonged retention in the intima, and ease of oxidation makes D-LDL particularly susceptible to the oxidative stress of the artery wall. Additionally, once mildly oxidized, D-LDL may be more atherogenic, with an enhanced capacity to stimulate monocyte chemotaxis and adhesion compared with B-LDL.28 Endothelial cells and monocytes/macrophages are an important source of oxidant stress in the artery wall. Monocytes and macrophages are present in the intima in very early stages of lesion formation, and both endothelial cells and monocyte/macrophages are capable of oxidizing LDL, at least in vitro.293031

These findings have particular relevance to individuals with diabetes. Preliminary data suggest that LDL from individuals with insulin-dependent diabetes mellitus and NIDDM is more susceptible to oxidation.3233 If, as in nondiabetics, D-LDL from NIDDM patients is more susceptible to oxidation than B-LDL, the greater prevalence of D-LDL in NIDDM compared with nondiabetics3435 may partially explain the enhanced susceptibility of LDL from individuals with NIDDM. In addition to the predominance of D-LDL, lipoproteins from individuals with diabetes may be altered in other ways, such as by glycation and advanced glycation end product formation, which would increase their risk for oxidation.3637 Monocytes from diabetic individuals may also have increased production of free radicals and enhanced ability to oxidize LDL.38 This may be related to their level of hyperglycemia or to the hyperlipidemia that is frequently present in these individuals.3940 Thus, LDL from diabetic subjects may be both more susceptible to oxidation and exposed to more oxidant stress from cells present in the artery wall.

Previous work by our laboratory41 has demonstrated that while vitamin E supplementation in nondiabetic individuals provides substantial protection to unfractionated LDL and B-LDL, it only modestly inhibits the susceptibility of D-LDL to oxidation. In these same individuals, the addition of MUFAs to the diet led to D-LDL that was substantially more resistant to oxidation. Supplementation with probucol, a potent antioxidant, markedly decreases LDL susceptibility to oxidation,42 although its effect alone or in combination with an MUFA-enriched diet on D-LDL has not been evaluated. Although vitamin E supplementation in NIDDM subjects provides a similar degree of protection to LDL as it does for nondiabetics,43 D-LDL is still readily oxidized. In view of the limited extent of antioxidant protection of D-LDL provided by vitamin E and the increased prevalence of this LDL fraction in NIDDM, we thought that interventions with more potent antioxidants and antioxidants in combination with MUFA-enriched diets may be needed in this high-risk group. Additionally, the effect of antioxidant supplementation on monocyte production of reactive oxygen species is also unknown. Therefore, this study evaluated in individuals with NIDDM the effects of supplementation with vitamin E or probucol alone or in combination with a diet enriched in MUFA on the susceptibility of LDL and LDL subfractions to oxidation and monocyte superoxide production.

Methods

Participants

Twelve NIDDM patients (4 women and 8 men) aged 37 to 69 years were recruited from endocrinology clinics at the Veterans Administration Hospital in San Diego. The duration of diabetes in the study group was 3 to 30 (mean, 10.5) years, and no subjects had renal insufficiency. All subjects were without acute medical problems and none took lipid-lowering medications or antioxidant supplementation. Hyperglycemia was controlled through the use of diet, oral agents, or insulin; at study entry subjects had fasting glucose levels ranging from 117 to 250 mg/dL and HbA1c levels from 5.5% to 10.4%. No changes in the management of hyperglycemia were instituted during the study. The study was approved by the human studies committee of the University of California, San Diego, and was conducted in the outpatient facilities of the University of California–San Diego, General Clinical Research Center.

Study Design

The 12 subjects were randomized to receive either all-rac–α-tocopherol (vitamin E) 1600 IU/d (Hoffmann–La Roche Research Institute) or probucol 1000 mg/d (Merrell Dow Research Institute) for 4 months. After 3 months, all subjects also began receiving foods enriched in oleic acid for the final 4 weeks of the 4-month study. An oleate-enriched variant of sunflower oil (Trisun 80) provided by SVO Enterprises was incorporated into various foods.41 Oleate accounts for >80% of the total fatty acids in Trisun 80. Diets were prepared by the General Clinical Research Center Nutrition Unit and provided ≈40% of calories as fat, 45% as carbohydrates, and 15% as protein. The percent of fat calories that were in the form of MUFAs, polyunsaturated fatty acids, or saturated fat were approximately 69%, 12%, and 19%, respectively. Baked foods were based on recipes formulated by the Clinical Research Center Nutrition Unit as were additional foods (tuna/chicken/pasta salad, fruits, granola, etc). Samples of each food item were analyzed for fat composition by SVO Enterprises and confirmed for composition accuracy. Based on food preferences, a 7-day cycle of meals was selected from the list of food items and prepared individually for each subject. Initial daily caloric intake for each individual was calculated from estimates of energy requirements that were based on 3-day food records, activity levels, and standard nomogram values44 for each individual. Daily dietary records were reviewed weekly by a registered dietitian to ensure adherence and to monitor acceptability. Total daily energy intake for each individual was adjusted weekly as needed to maintain body weight.

Participants picked up their prepared diets on Monday, Wednesday, and Friday of each week for the 4-week study period. Unused portions of diet were returned at each visit. Each participant was allowed to ingest their daily diet according to their own schedule, although the entire day's allocation was to be ingested by bedtime. Subjects were instructed to refrigerate perishable foods at all times when not in use.

Preparation of LDL

Fasting blood samples were obtained from each subject at baseline, at the end of 3 months of antioxidant supplementation, and at completion of the 4-week dietary period. Plasma was separated from blood that had been collected in tubes containing EDTA (4.0 mmol/L) and placed immediately on ice. LDL was isolated by sequential ultracentrifugation43 and dialyzed extensively against PBS containing 0.27 mmol/L EDTA (PBS-EDTA). Plasma was also divided into two equal aliquots and separated into buoyant and dense fractions of LDL by performing two sequential ultracentrifugation spins with each portion of plasma. Density spins of d=1.022 g/mL and d=1.032 g/mL were used to isolate B-LDL and d=1.040 g/mL and d=1.054 g/mL for D-LDL. Samples were brought to the appropriate density by the addition of NaBr and were centrifuged in 6-mL Ultra-Clear tubes in a Beckman 50.3 fixed-angle rotor at 40 000 rpm for 20 hours at 10°C.4143 These density ranges provide significantly more D-LDL than B-LDL in plasma.41

Measurement of LDL conjugated diene formation during copper-mediated oxidation was performed immediately after isolation, as described below. LDL samples were subsequently stored at 4°C in the dark, and all other studies were completed within 1 week of isolation. For all oxidation assays, LDL samples were dialyzed against several changes of PBS over 20 hours in the dark to remove all EDTA before use in experiments.

Vitamin E Content

α-Tocopherol was measured by using high-performance liquid chromatography,43 and α-tocopherol acetate was prepared in 100% ethanol and used as an extraction internal standard and for standard curve preparation. Actual concentrations of α-tocopherol were determined by measuring the absorbance of prepared solutions and calculating concentrations based on known spectral data. Calculations were determined from a standard curve of peak area ratios of sample to internal standard.

Oxidation of LDL

The formation of conjugated dienes was measured by incubating 75 μg LDL protein with 2.5 μmol/L copper sulfate (Cu2+) in 1 mL PBS at 30°C. The absorbance at 234 nm was measured continuously in a Uvikon 810 spectrophotometer.45 For data presentation, the first derivative of the rapid phase of oxidation was calculated (slope), and its intercept with the x axis (lag time) was determined.45 Copper-mediated oxidation appears largely dependent on the presence of preformed lipid peroxides.46

Fatty Acid Composition

Lipids from LDL and LDL subfractions were extracted by using a modification of the method of Folch et al.47 The fatty acids were transmethylated and analyzed in a Varian gas chromatograph (model 3700) equipped with a column of 10% Silar 5CP on a Gas Chrom QII, 100/120 mesh. A 15:0 internal standard (pentadecanoic acid) was added to each sample before extraction, and calculations of fatty acid amounts were determined from peak area ratios of sample to internal standard.

Monocyte Isolation and Cell Assays

Human monocytes were isolated by using a modification of the method of Boyum.48 Briefly, mononuclear cells were recovered from the interface formed during ultracentrifugation of the white blood cell pellet on a Histopaque gradient. Cells were then washed in PBS containing 0.02% EDTA-PBS, resuspended in autologous serum (10%) and RPMI 1640 medium, and plated. After a 2-hour incubation at 37°C, nonadherent cells were washed off, leaving a highly enriched monocyte preparation. The remaining adherent cells were then gently released by using 0.2% EDTA-PBS, washed with RPMI 1640, and recounted. These preparations were >90% monocyte pure. A portion of these cells was immediately used for measurement of superoxide anion production by the superoxide dismutase–inhibitable reduction of cytochrome c.49 Cytochrome c (80 mmol/L) was added with and without phorbol myristate acetate at a concentration of 5 ng/mL to monocytes plated at 5×105 cells/well for 60 minutes at 37°C, and the absorbance of the supernatant was measured at 550 nm. Identical reactions were also performed with superoxide dismutase (300 U/mL) included to determine the superoxide dismutase–inhibitable cytochrome c reduction.

Lp(a) Isolation

Plasma was adjusted with 0.2 g NaBr/mL and overlaid with 10 mL NaBr at d=1.030. Samples were centrifuged in an SW41 rotor at 38 000 rpm for 12 hours at 8°C. The Lp(a) band was removed in a 1-mL volume ≈9 mm from the infranate.50

Statistical Methods

Statistical comparisons within treatment groups were performed by using repeated-measures ANOVA with subsequent post hoc paired comparisons. Pearson correlation coefficients were determined to evaluate correlations between selected variables.

Results

Eleven of 12 participants completed the trial. One subject from the vitamin E group removed herself from the study for personal reasons prior to the initiation of the diet-intervention phase. Pill counting revealed that >95% of distributed pills were taken. Similarly, compliance with the dietary phase of the study was high, with >90% of the provided food ingested as determined by food records and returned portions.

Weights did not change significantly in either group during the study. There was also no significant change in fasting glucose or HbA1c levels in either group (Table 1⇓), demonstrating that our efforts to maintain stable glucose control were successful. Mean values for plasma lipids and lipoproteins during the study are shown in Table 2⇓. Consistent with most reported studies,5152 there was no significant change in lipid values during supplementation with vitamin E alone. After the addition of the MUFA-enriched diet, TC and LDL-C decreased significantly compared with values on vitamin E alone. In contrast, TC, LDL-C, and HDL-C levels all decreased with probucol alone, although only the decrease in HDL-C reached significance (P<.05). The addition of an MUFA-enriched diet led to further reductions in TC and LDL-C. The major change in HDL-C values occurred during the probucol-only phase, with no significant further decrease occurring after the addition of the MUFA diet.

Levels of vitamin E in LDL are shown in Table 3⇓. Vitamin E increased during the study in both the vitamin E–supplemented group and after MUFA supplementation in the probucol-supplemented group. In the vitamin E group a significant increase occurred during the first 3 months of vitamin E supplementation, with a slightly smaller additional increase occurring after the initiation of the MUFA diet. The relative increase in vitamin E content was similar in all LDL fractions, although absolute levels were greatest in B-LDL. The vitamin E/LDL lipid ratio, however, did not differ significantly between LDL subfractions (data not shown). In contrast, in the probucol group, vitamin E increased only during the MUFA diet phase. These data suggest that the addition of MUFAs may have enhanced the LDL levels of vitamin E in both groups. Probucol levels in LDL increased to 6 to 9 mol/1 mol LDL by 3 months of supplementation and only increased modestly after this time (Figure⇓). Again, D-LDL was less enriched on a mole/mole basis than was the B-LDL or unfractionated LDL.

Line plot shows probucol levels in unfractionated LDL (□), D-LDL (•), and B-LDL (▪). LDL was isolated at the indicated times from individuals who had received probucol 1 g/d for 4 months and an MUFA-enriched diet for the final month. Values are mean±SE.

There was no change in fatty acid composition in the LDL from either group during the antioxidant supplementation phase, and presupplementation levels were comparable with those of nondiabetics.94153 After the MUFA supplementation phase began, significant increases in 18:1 and significant decreases in 18:2 occurred (Table 4⇓). There were also modest decreases in 16:0, 16:1, 18:0, and 20:4 (data not shown). The extent of change in these fatty acids was similar in LDL and LDL subfractions and similar to the changes in LDL composition that have been reported after MUFA supplementation.9104153

LDL and LDL Subfraction Fatty Acid Content at Baseline and After Antioxidant and Diet Interventions

The effects of vitamin E or probucol supplementation alone and in combination with MUFA on LDL oxidation are shown in Table 5⇓. Prior to supplementation, the lag time of D-LDL and unfractionated LDL were similar and significantly shorter than that of the B-LDL. Vitamin E significantly increased the lag time substantially in all classes of LDL, although the lag time of D-LDL remained shorter than that of either B-LDL or LDL. MUFA supplementation did not further increase the lag times of unfractionated LDL or B-LDL, but it increased the lag time of D-LDL by over 70 minutes (P<.02). Supplementation with probucol alone increased lag times of LDL and LDL subfractions by three- to fourfold compared with presupplementation levels. Lag times of probucol-enriched LDL were substantially greater than those achieved with vitamin E enrichment (P<.05). However, similar to what occurred in the vitamin E group, the average increase in lag time as a result of probucol enrichment of D-LDL was 104 minutes less than that gained by probucol enrichment of unfractionated LDL and 411 minutes less than that gained by probucol enrichment of B-LDL. In contrast, the addition of an MUFA-enriched diet had its greatest effect on D-LDL, with a further 18% increase in lag time, compared with 9% and 2% increases for unfractionated LDL and B-LDL, respectively. The rate or propagation of LDL oxidation (as measured by the slope of the conjugated diene curve) was also reduced by supplementation with vitamin E or probucol. Although these changes in the rate of LDL oxidation were consistent, they were modest in size and achieved significance only for unfractionated LDL. Similarly, the effect of MUFA supplementation on the rate of oxidation was modest.

To evaluate the effect of antioxidant supplementation on monocyte production of free radicals, measurements of basal and phorbol ester–stimulated production of superoxide anion (in nanomoles per milligram cell protein) were determined in monocytes from each subject at each visit. Basal (vitamin E: 313±150, 395±95, and 357±135; probucol: 325±114, 389±24, and 345±88) and phorbol myristate acetate–stimulated (vitamin E: 894±278, 652±127, and 764±121; probucol: 822±200, 686±165, and 829±133) levels of superoxide anion did not change significantly over the course of the study and did not differ between groups.

The inhibitory effect of vitamin E supplementation on LDL oxidation was borne out by the strong positive correlation between LDL vitamin E content and lag time for all LDL fractions (r=.82 for LDL, r=.78 for B-LDL, and r=.79 for D-LDL, P<.05). Similarly, the content of probucol in LDL was positively correlated with the LDL lag time in the probucol-supplemented group (r=.97 for LDL, r=.91 for B-LDL, and r=.97 for D-LDL, P<.05). Excluding the samples from the baseline visit in the probucol group (which contained no probucol) did not significantly alter the results. To evaluate whether glucose control alone may influence the lag time of LDL oxidation, comparisons of baseline HbA1c values to LDL lag times were made for each LDL fraction. Although there was an inverse relationship between HbA1c and lag time for all LDL fractions, which suggests that hyperglycemia may enhance LDL oxidation, none reached significance (r=−.23 for LDL, r=−.44 for B-LDL, and r=−.44 for D-LDL). LDL MUFA and polyunsaturated fatty acid content also correlated with lag times.53 The correlation between the percent of 18:1 and 18:2 in LDL with lag time from all subjects over the course of the study was strong for all LDL fractions but greatest in D-LDL (r=.70 for 18:1 and r=−.59 for 18:2, P<.5). This is consistent with the above findings that MUFAs were most effective in inhibiting oxidation in D-LDL.

Discussion

The major goals of this study were twofold: to evaluate the effects of supplementation of vitamin E or probucol alone or in combination with an MUFA-enriched diet on LDL and LDL subfraction susceptibility to oxidation and to determine the effects of these combinations on cellular prooxidant activity. We felt it was particularly relevant to evaluate these interventions in NIDDM subjects because their LDL is frequently more enriched in D-LDL particles3435 and is more susceptible to oxidation3233 and because they reportedly have monocytes that overproduce superoxide anion.3840

We were unable to demonstrate a significant reduction in superoxide anion production from isolated monocytes at any time during the study. This was true both for unstimulated and phorbol ester–stimulated monocytes. This was not simply a result of limited uptake by monocytes of vitamin E or probucol. After 3 months of vitamin E supplementation, monocyte vitamin E content was four- to fivefold higher than pretreatment levels. Similarly, probucol content increased from undetectable levels to 23.5±9.8 ng/mg cell protein in monocytes isolated from subjects receiving probucol. However, there may be other antioxidants, such as glutathione or N-acetyl-l-cysteine, which are more effective in decreasing the cellular production of reactive oxygen species. These findings also do not rule out the possibility that vitamin E and probucol have effects on other cell functions, such as cell differentiation, or that extracellular antioxidants may temper the oxidant stress of the respiratory burst; they do suggest, however, that antioxidant supplementation may have a limited effect on cellular prooxidant activity as measured by superoxide anion production.

Supplementation with these antioxidants did have significant effects on LDL antioxidant content and LDL susceptibility to oxidation. After supplementation D-LDL was less enriched with both vitamin E and probucol than was B-LDL. Interestingly, the addition of MUFA-enriched diets led to increased vitamin E content in LDL from both the vitamin E and probucol groups. This raises the possibility that enrichment of LDL with 18:1 (and reductions in 18:2) may reduce in vivo vitamin E loss to oxidation. This is supported by the presence of consistent elevations of vitamin E in all LDL fractions from both groups during the MUFA phase of the study. It is unlikely that these changes in LDL vitamin E were solely a result of increased vitamin E intake in the diet. The vitamin E content in Trisun oil (0.54 mg/g) is equal to or lower than that present in most polyunsaturated oils. Foods enriched with Trisun oil would provide at most an additional 50 to 60 mg of vitamin E per day, which might slightly increase LDL levels in non–vitamin E–supplemented subjects, but not to the extent seen in the probucol group. Moreover, this amount of vitamin E would not affect levels in those already receiving 1600 IU/d. It is possible that in diabetic subjects, who may have increased rates of in vivo lipid peroxidation and consequently greater rates of vitamin E utilization, the vitamin E sparing effect of an MUFA-enriched diet is particularly prominent. Probucol levels in LDL also increased modestly in MUFA-supplemented LDL samples. However, in contrast to the relatively rapid rise in vitamin E levels that occurred in plasma and LDL with vitamin E supplementation, plasma and LDL probucol levels rose gradually over months, and the higher levels present during the MUFA supplementation phase may have resulted not only from sparing of probucol but in part from ongoing probucol supplementation.42 Although this may account in part for the enhanced lag time found in the probucol-treated group after the MUFA supplementation phase, several lines of evidence suggest that MUFA supplementation may have had independent beneficial effects. The changes in probucol levels in LDL from month 3 to month 4 were very modest (and nonsignificant). This is supported by the modest percent of change in lag time from month 3 to 4 (r2=.09), which is explained by the minor change in the probucol concentration. Additionally, the ability of probucol to reduce LDL oxidation (as measured by using thiobarbituric acid–reactive substances) essentially plateaus after 2 to 3 months of supplementation.42

LDL enriched in vitamin E was less susceptible to oxidation, although the protection provided to D-LDL was less than that provided to B-LDL and unfractionated LDL. Both these findings are consistent with previous reports.4143 The addition of an MUFA-enriched diet further increased the lag time of only D-LDL. A similar pattern was seen in the group supplemented with probucol. Although probucol provided substantially better protection for D-LDL than did vitamin E, a similar pattern was present, as D-LDL remained the fraction most readily susceptible to oxidation even after enrichment with this potent antioxidant. However, the addition of an MUFA-enriched diet again appeared to reduce the susceptibility of D-LDL to oxidation to a greater extent than that of other LDL fractions.

At the end of the study two subjects from each group had plasma Lp(a) levels sufficiently high so that these particles could also be isolated and exposed to oxidative stress and compared with Lp(a) particles isolated from two control subjects. Vitamin E together with MUFA supplementation increased the lag time of Lp(a) oxidation by nearly twofold (to 195±9.9 minutes), while probucol and MUFA supplementation lengthened the lag time by more than fourfold (to 496±66 minutes) compared with Lp(a) from untreated subjects (92±17 minutes). Thus, the percent increase in the lag time of Lp(a) oxidation resulting from these antioxidant and dietary manipulations was similar to that obtained for LDL, although the absolute lag times were less. Lp(a) levels have been associated with coronary artery disease in numerous studies (for review, see Reference 54). The exact mechanism of this association is unknown, although most proposed explanations have focused on prothrombotic properties of Lp(a).5556 However, oxidation of Lp(a) particles may also contribute to their atherogenicity, as they very likely have many of the proatherogenic properties of oxidized LDL, including generation of chemotactic molecules,3467 impairment of endothelial function,5758 and uptake by macrophages via scavenger receptors and possibly as well as through phagocytosis.59 Although the number of Lp(a) samples is small, these results are consistent with several reports6061 that suggest reduced Lp(a) susceptibility to oxidation may be an additional benefit of antioxidant supplementation.

These findings point out the markedly different effects that antioxidants and diet may have on different LDL fractions, which may be particularly important when designing antioxidant intervention trials in groups prone to have increased amounts of D-LDL, such as in NIDDM. Additionally, this confirms our findings in nondiabetic subjects41 that fatty acid composition is particularly important in determining D-LDL susceptibility to oxidation. The relatively strong correlation between LDL fatty acid composition (18:1 and 18:2) and D-LDL lag times supports this relationship. Why MUFA-enriched diets would be particularly effective in reducing susceptibility of D-LDL to oxidation is unclear. One can speculate that since D-LDL has a higher 18:2 to 18:1 ratio,41 it benefits the most from a diet that lowers 18:2 and raises 18:1. It has also been proposed that the phospholipid-rich surface layer of D-LDL is particularly susceptible to oxidation62 and that its structural organization is uniquely permissive to the transmission of free radicals to the core lipids.63 This combination would certainly make D-LDL particularly susceptible to oxidation. Surprisingly, during oxidation the 18:1 content within the phospholipid fraction of LDL is relatively preserved, as if it is uniquely protected from or resistant to oxidation while in the surface layer.53 Greater enrichment of the phospholipid layer with 18:1 (and depletion of 18:2) through MUFA-enriched diets may further reduce phospholipid fatty acid oxidation and slow LDL oxidation. Presumably, this effect would be greatest in D-LDL. Alternatively, vitamin E may be more oxidatively labile in D-LDL,64 thereby providing less effective protection against oxidation. Increased vitamin E lability may be influenced by factors such as lipoprotein composition and thus may be reduced in D-LDL particles enriched in MUFA. Further studies of the mechanisms underlying the effects of MUFA enrichment on D-LDL oxidation are clearly needed. Nevertheless, these findings may have important therapeutic implications. In the present study, the amount of D-LDL in plasma was greater than that of B-LDL. Furthermore, oxidation of D-LDL not only occurs more readily than that of B-LDL, but once oxidized it may take on particularly atherogenic properties, such as the ability to enhance monocyte chemotaxis, migration, and adherence.2865 If future studies continue to implicate D-LDL as an important particle in the atherogenic process, therapeutic strategies to reduce its oxidation will be needed. This may be particularly relevant in individuals with an increased prevalence of small dense LDL, such as occurs in NIDDM patients.

It is currently agreed that in nearly all NIDDM patients, reducing dietary saturated fat helps lower plasma cholesterol levels and reduces the risk for coronary heart disease. There is, however, some controversy as to whether dietary saturated fat should be replaced by carbohydrates or MUFAs in NIDDM. Although it is not yet clear why MUFA-enriched diets are particularly effective in inhibiting D-LDL oxidation, this effect may provide an additional reason to consider using MUFA-enriched diets in NIDDM patients.

Selected Abbreviations and Acronyms

B-LDL

=

buoyant LDL

D-LDL

=

small, dense LDL

HbA1c

=

hemoglobin A1c

HDL-C

=

HDL cholesterol

LDL-C

=

LDL cholesterol

Lp(a)

=

lipoprotein(a)

MUFA

=

monounsaturated fatty acid

NIDDM

=

non–insulin-dependent diabetes mellitus

PBS

=

phosphate-buffered saline

TC

=

total cholesterol

Acknowledgments

This work was supported by grant HL-14197 from the National Heart, Lung, and Blood Institute (SCOR), and GCRC Program M01 RR00827 of the National Center for Research Resources, National Institutes of Health. We thank SVO Enterprises for supplying Trisun oil, Marion Merrell Dow Research Institute for supplying probucol capsules, and Hoffman–La Roche Research Institute for supplying vitamin E and for additional financial support. We thank Dr Joseph Witztum for his advice and comments regarding the manuscript; Haven Webb and Elizabeth Miller for important assistance in the conduct of these studies; Annie Durning-Canty and Eva Brzezinski from the General Clinical Research Center Nutrition Unit for excellent assistance in diet preparation and nutrient analysis; and Lisa Gallo for assistance in manuscript preparation.

Reaven PD, Grasse BJ, Tribble DL. Effects of linoleate-rich and oleate-rich diets in combination with α-tocopherol on the susceptibility of LDL and LDL subfractions to oxidative modification in humans. Arterioscler Thromb.1993;14:557-566.